Multivariate morphometric analysis of sect. Petrorhagia subsect. Saxifragae in Greece, with the description of a new from SE Peloponnisos

PANAYIOTIS TRIGAS1*, KONSTANTINOS KOUGIOUMOUTZIS1, AIKATERINI ERMIDOY1 & ELEFTHERIOS KALPOUTZAKIS2 1Laboratory of Systematic Botany, Department of Crop Science, Agricultural University of Athens, Iera Odos 75, 118 55, Athens, Greece; e-mail: [email protected] 2Division of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, National and Kapodistrian University of Athens, 157 71 Athens, Greece *author for correspondence

Abstract

The intra-generic relationships within the intriguing Petrorhagia are currently debated, as the genus is underrepresented in recent taxonomic studies. In this study, we investigate the relationships among the species of Petrorhagia sect. Petrorhagia subsect. Saxifragae occurring in Greece. Based on a combination of ordination methods and discriminant analyses of 36 macromorphological characters, five species are recognized in Greece. The analyses showed that all currently known species from Greece (P. fasciculata, P. graminea, P. phthiotica and P. saxifraga) represent distinct taxonomic entities. According to our results, two recently collected populations from SE Peloponnisos represent a fifth, new Petrorhagia species, which is first described and illustrated here as P. laconica. It is related to the widespread P. saxifraga and the Peloponnesian endemic P. graminea, but clearly distinct from both species by a combination of morphological characters, including indumentums, , calyx, capsule and seed characters.

Key words: , Greek flora, Morphology, Petrorhagia laconica

Introduction

Caryophyllaceae include approximately 3000 species, distributed in about 100 genera (Hernández- Ledesma et al. 2015). It has a primarily north-temperate distribution with a diversity center in the eastern Mediterranean and Irano-Turanian regions, while presence in the tropics and the southern hemisphere is limited and mostly at higher elevations (Bittrich 1993, Greenberg & Donoghue 2011, Rabeler & Hartman 2005, Hernández-Ledesma et al. 2015). The phylogenetic relationships within Caryophyllaceae is a field of active research (Fior et al. 2006, Harbaugh et al. 2010, Greenberg & Donoghue 2011, Hernández-Ledesma et al. 2015, etc.) and genera delimitation has been altered in some cases, reflecting the results of recent molecular studies (e.g. Dillenberger & Kadereit 2014). Petrorhagia (Seringe in Candolle 1824: 354) Link (1831: 235) (Caryophylloideae, Caryophylleae) is a small genus of ca. 33 species, distributed in the Mediterranean area, Europe and western Asia (from the Canary Islands east to Kashmir), with a diversity center in Greece and Turkey (Ball & Heywood 1964, Georgiou 1997). In the past, it was placed in different genera like Linnaeus (1753: 409), Tunica Scopoli (1772: 298), Kohlrauschia Kunth (1838: 108) and Linnaeus (1753: 406). Evidence from both traditional and phylogenetic systematics, support its recognition as a distinct taxon at the genus rank, which clusters as sister to a clade including Dianthus Linnaeus (1753: 409) and Velezia Linnaeus (1753: 332) (Ball & Heywood 1964, Harbaugh et al. 2010, Greenberg & Donoghue 2011, Pirani et al. 2014, Hernández-Ledesma et al. 2015, Hilooğlu et al. 2016). Recent data on the infrageneric classification of Petrorhagia is still scarce, as the genus is rather under-sampled. The available data (Greenberg & Donoghue 2011, Hilooğlu et al. 2016) are fragmentary and do not allow a reliable infrageneric classification. As a result, most recent treatments of the genus follow the monograph of Ball & Heywood (1964) that recognizes five sections within Petrorhagia, distinguished by and seed characters, life-cycle, leaf venation and the presence/absence of epicalyx bracts. Out of the five sections, sect. Petrorhagia includes ten species (seven of them present in Greece and five endemic to the country) distributed into two subsections: subsect. Saxifragae Ball & Heywood (1964: 148) and subsect. Thessalae Ball & Heywood (1964: 156). The two subsections are distinguished by stem branching (much branched vs. simple or with few branches), inflorescence ( solitary or fasciculate vs. capitates) and bract (lanceolate or ovate, membranous, 1-veined vs. broadly ovate or suborbicular, membranous and 1- veined or brown-scarious and many-veined) morphological characters. In May 2014, during field work in south-eastern Peloponnisos, we detected a population of an interesting Petrorhagia species, growing on coastal sand-dunes, an unusual habitat for the members of the genus. Careful examination of the collected specimens revealed that from SE Peloponnisos belong to Petrorhagia sect. Petrorhagia, and they resemble P. saxifraga (Linnaeus 1753: 413) Link (1831: 235), a widely distributed species in central and southern Europe and south- western Asia. After a detailed morphological study, it became apparent that plants from SE Peloponnisos are clearly distinct from P. saxifraga, as well as from all known Petrorhagia species and they belong to a new, undescribed species. Dr. E. Kalpoutzakis had also found the same species in May 2013 in another locality of SE Peloponnisos. The morphological characters of the new species from SE Peloponissos, described here as Petrorhagia laconica, support its classification in subsect. Saxifragae. The of the subsect. Thessalae is rather clear compared to that of the subsection Saxifragae. The species belonging to the latter are notorious for their taxonomic complexity; their confusing taxonomy seems to be due to the rather few species-delimitating characters. Species delimitation within Petrorhagia sect. Petrorhagia subsect. Saxifragae has never been studied in detail. All species of subsect. Saxifragae, except P. riphaea (Pau & Font Quer) Ball & Heywood (1964: 156) that is the only species of this group distributed in Africa (Morocco), are distributed in Greece. Thus, the scope of this study is manifold; it aims to resolve the apparent ambiguities within subsection Saxifragae and to evaluate the taxonomic validity of the two recently discovered deviating Petrorhagia populations. More specifically, we aim: (a) to clarify the patterns of morphological variation within Petrorhagia sect. Petrorhagia subsect. Saxifragae in Greece, (b) to disclose the level of morphological differentiation for recognized species, (c) to indicate the most informative characters for the identification of the species, and (d) to describe the newly discovered species.

Material and methods

Plant Material Data for the multivariate analyses were recorded from herbarium specimens deposited in UPA and ATH (acronyms follow Thiers 2015) belonging to Petrorhagia fasciculata, P. graminea, P. phthiotica and P. saxifraga. We collected several specimens of P. laconica in the field. The specimens included in the analyses were selected so as to represent the taxa’s entire distribution range in Greece, as well as the morphological variation in each species. Only well-preserved and intact specimens were considered in the analyses. The total number of herbarium specimens included in the analyses was 97. A list of the examined specimens is provided in Table S1. Each specimen was preliminary identified as one of the five aforementioned species. Species identification and nomenclature are according to Ball & Heywood (1964), Georgiou (1997) and Dimopoulos et al. (2013).

Characters recorded Twenty-seven quantitative and 9 qualitative (binary) morphological characters were scored for the herbarium specimens included in our analyses (Table 1). The selected characters represent all those previously quoted as diagnostic and are the ones most commonly used in the latest comprehensive monographs, regional and local floras (Ball & Heywood 1964, Coode & Cullen 1967, Georgiou 1997). All the characters in the herbarium specimens were measured under a stereomicroscope using a ruler with the precision of 0.5 mm.

Morphometric Analyses Basic statistical parameters (mean, minimum and maximum value, standard deviation, 5th and 95th percentiles) were calculated for each species included in the analyses. We used a combination of ordination methods and discriminant analyses in the morphometric analyses of the specimens (Marhold 2011). We first tested whether any character did not have a normal distribution using the Shapiro-Wilk statistic and we log10-transformed any characters that deviated from normality. As a second step, we performed several stepwise canonical and classificatory Discriminant Analyses (DAs) in order to: i) identify the variables having the highest potential as diagnostic characters and ii) test the discrimination rules’ effectiveness by using the Leave-One-Out cross-validation (LOO). The inclusion and the exclusion criterion’s value in the stepwise DAs was set at F = 0.05 and F = 0.1, respectively. Wilk’s lambda was used to assess each variable’s and Discriminant Function’s (DF) significance. The Maximum Chance Criterion (MCC) and the Proportional Chance Criterion (PCC) were used to determine whether the prediction equation was better than random chance (Huberty & Olejnik 2006). The DAs were checked for outliers. Finally, we examined the morphological variation in our dataset in relation to the species boundaries by analyzing the characters’ ranges using core functions in the R computing environment (R Core Team 2015). All the analyses were carried out using IBM SPSS 24. The stepwise discriminant analysis plots, as well as the violin boxplots were visualized via the ggplot2 (Wickham, 2009) package in the R computing environment. The Maximum and Proportional Chance criteria were ran under the Zclass algorithm kindly provided by John D. Morris (Florida Atlantic University).

Results

Characters scored The basic statistical parameters for all species are given in Table S2. The characters’ morphological variation is graphically depicted in Figure S1, respectively. The scored characters’ distribution departed from normality; therefore, all the characters were log10-transformed prior to the analyses. After the logarithmic transformation, all the characters were normally distributed.

Multivariate analyses We performed several canonical and discriminant analyses in order to find the most important characters separating the a priori defined groups, as well as to test the potential success of such separations. The DA clearly discriminates the five Petrorhagia species. Sixteen of the characters emerged as statistically significant (Table 2), with LOO showing a 100% correct classification, being significantly better than random chance for both the PCC and MCC (P < 0.001, Table 3). These characters showed no to little overlap across the five species (Fig. 1). The first (DF1), the second (DF2), the third (DF3) and the fourth (DF4) discriminant function explain 86.6%, 11.5%, 1.3% and 0.6% of the total variation, respectively (Table 2, Figs. 2, S2). Among the morphological characters used, the presence and the type of the indumentums are the most informative characters for species distinction in Petrorhagia sect. Petrorhagia subsect. Saxifragae. The most discriminative function (DF1) is primarily influenced by the presence of eglandular hairs at the middle part of the stem (a9). The presence of glandular hairs on calyx (a34) and eglandular hairs at the lower part of the stem (a8) have also high discriminant validity in DF1 (Table 2). The presence of eglandular hairs on calyx (a35) and glandular hairs at the middle/upper part of the stem (a6/a7) are the characters loading most heavily on DF2 and DF3, respectively (Table 2). Undoubtedly, the five Petrorhagia species included in the analyses are clearly distinct from one another (Table 3, Figs. 1-2).

Discussion

Multivariate morphometrics provide a powerful mean in order to assess the variation patterns at various taxonomic levels and is especially useful in disentangling the boundaries between complex taxa (e.g., Kougioumoutzis et al. 2015), as well as in unveiling the most informative and discriminating characters that differentiate among them (Marhold 2011). In the present study, the application of multivariate morphometrics resulted in important conclusions with taxonomic consequences. More specifically, the results of morphometric analyses enabled the delimitation of five well-separated species and the elucidation of the identity of the deviating Petrorhagia populations from SE Peloponnese: the latter constitute a new, distinct and rather rare taxon, herein described as Petrorhagia laconica. Petrorhagia sect. Petrorhagia subsect. Saxifragae is a taxonomically complicated group with several overlapping characters. Our morphometric analyses, however, showed that all species are clearly distinguished each other. Their distinction can be achieved even without using seed morphological characters, an important diagnostic feature in the genus Petrorhagia. The latter were not included in the morphometric analyses, as most specimens used were not in fruit. The presence and type of hairs on stem and calyx have emerged as important diagnostic characters. The presence of uniformly eglandular hairy stems throughout their length is a unique feature of P. laconica. P. fasciculata and P. phthiotica are the only species with uniformly glandular hairs on stems, while the stems of P. graminea are covered by a mixture of glandular and eglandular hairs, at least at base. All examined specimens of P. saxifraga had glabrous stems. Furthermore, P. laconica is the only species with uniformly glandular hairy calyx. The calyx of P. graminea is predominantly covered by eglandular hairs, often mixed with few glandular hairs. All examined specimens of P. fasciculata had eglandular hairy calyces, while all calyces in P. saxifraga and P. phthiotica were glabrous. Length of the inflorescence, number of lateral branches and flowers per flowering stem, width of lower leaves and the ratio pedicel/calyx length further differentiate P. laconica from all other species of sect. Saxifragae (Table S2, Figure S1). Moreover, P. laconica, together with P. saxifraga, are the only species with calyces enclosed by epicalyx bracts; in the specimens of all other species (including P. fasciculata) there are not bracts enclosing the calyx. The small and the large number of flowers per terminal inflorescence unit easily distinguish P. fasciculata from all other species. P. graminea and P. saxifraga are the only species with 3-veined and they also have the largest petals. The 3-veined leaves and the short stems further differentiate P. phthiotica from all other members of sect. Saxifragae. The results of our analyses have revealed a diagnostic character not previously used in the genus Petrorhagia. Although the length of leaves is similar in all species examined, leaf width varies among species. P. saxifraga and P. laconica have narrow basal leaves compared to the other species. Furthermore, cauline leaves are diminishing in size towards apex in all species, but diminishing in leaf length only, while leaf width remains stable, has been observed only in P. laconica (Table S2, Figure S1).

Taxonomy

Petrorhagia laconica Trigas, Kalpoutzakis & Kougioumoutzis, sp. nov. (Fig. 3)

Related to P. saxifraga, from which it differs in the following morphological characters: underground stock rooting at nodes; stems glaucous-green, procumbent to ascending or erect, simple or rarely with few [1–3 (–6)] racemose lateral branches at the upper 1/3, minutely pappilose- scabridulous except the glabrous 1–4 upper internodes; leaves rigid, closely appressed to stem; flowers solitary or rarely paired, 1 (–2) per flowering stem, rarely up to 6 and then inflorescence racemose; calyx minutely glandular pubescent; ribs obscurely 1-veined, purplish-brown; capsule 2.5–3 mm long, sparsely tuberculate at the lower half; seeds with flat, not thickened margins. Type:—GREECE. Peloponnisos: sand dunes ca. 3.5 km NW of Neapoli village, 2 m, 36o31.721’ N, 23o01.273’ E, 22 May 2017, Trigas 6311 (holotype ACA, isotype UPA).

Perennial herb with branched, slender, underground woody stock rooting at nodes, producing few to several flowering stems. Flowering stems glaucous-green, procumbent to ascending or erect, 7–40 cm long, green to purplish-brown, simple or rarely with few [1–3 (–6)] paniculate lateral branches at the upper 1/3, minutely pappilose-scabridulous except the glabrous 1–4 upper internodes; internodes markedly unequal, larger at the middle of the stem, diminishing in size towards base and apex. Leaves linear-subulate, acuminate to caudate, rigid, 1-veined, 5/4–1/3 as long as internodes, united at the base into a sheath 1–1.5 mm long, with a purplish-brown ring at the base; lower ones 7–19 × 0.5– 1 mm, minutely roughly papillose at margin, otherwise glabrous; cauline leaves closely appressed to stem, 3–16 × 0.5–1 mm, glabrous. Flowers solitary or rarely paired at apices of stems and branchlets, subtended by 4 hyaline, lanceolate, glabrous, straw-colored epicalyx bracts, sometimes purplish tinged at midrib. Calyx 3–6 mm long, cylindrical, minutely glandular pubescent; ribs obscurely 1- veined, purplish-brown; teeth 0.5–1 mm long, oblong-triangular, obtuse. Petals 4–8 × 1.5–3 mm, white, upper surface with three longitudinal purplish stripes at the base, lower surface purple veined, with glabrous claw not distinctly delimited from the emarginate lamina. Anthers white. Ovary ca. 1 mm long, oblong-ovoid; anthophore ca. 0.5 mm long, glabrous, green. Capsule 2.5–3 mm long, pale brown, ovoid, smooth or sparsely tuberculate at the lower half. Seeds 1.0–1.2 × 0.7–0.8 mm, ovate- oblong, with flat, not thickened margins, mucronate with a long, terminal mucro, reticulate on both surfaces, blackish-brown.

Etymology:—Species name refers to Laconia (also known as Lacedaemonia), a region that covers almost the entire SE part of the Peloponnisos, where the new species was collected. This name goes back to the historical times, when Laconia was the principal region of the Spartan state. Distribution:—Petrorhagia laconica is distributed in the Malea Peninsula in SE Peloponnisos. Two populations have been discovered so far; the first one is located at the Gulf of Neapoli, right across Elafonisos Island, while the second one was found close to Sykea village, ca. 25 km N-NW of the first population. It is the southernmost distributed species of Petrorhagia sect. Petrorhagia subsect. Saxifragae in Europe. Habitat and ecology:—Petrorhagia laconica is known to grow in two different habitats. In Neapoli, all individuals were observed to grow in the inner sand dune zone, usually surrounded by sclerophyllus shrubs, like Ceratonia siliqua Linnaeus (1753: 1026), Pistacia lentiscus Linnaeus (1753: 1026), Pyrus spinosa Forsskål (1775: 211), Smilax aspera Linnaeus (1753: 1028) and Anthyllis hermanniae Linnaeus (1753: 720). P. laconica grows in stabilized sand dunes among the shrubs, together with other sand dune adapted taxa, like Scirpoides holoschoenus (L.) Soják (1972: 127), Elytrigia juncea (L.) Nevski (1933: 17), Asphodelus ramosus Linnaeus (1753: 310), Centaurea sonchifolia Linnaeus (1753: 915), Linaria tenuis (Viv.) Sprengel (1825: 795) and Anthemis tomentosa Linnaeus (1753: 893). Close to Sykea village, Petrorhagia laconica grows in small remnant patches of macchie and phrygana formations that form a mosaic among the abundant olive groves. The population occupies a small area at 120–140 m a.s.l., on flysch. Shrub species composition of macchie is similar to that of the locus classicus, with Pistacia lentiscus, Pyrus spinosa, Olea europaea Linnaeus (1753: 8), Lavandula stoechas Linnaeus (1753: 573) and Phlomis fruticosa Linnaeus (1753: 584) being the predominant species. P. laconica usually grows in small patches among the shrubs, together with Sarcopoterium spinosum (L.) Spach (1846: 43), Ballota acetabulosa (L.) Bentham (1834: 595), Cistus salviifolius Linnaeus (1753: 524), Globularia alypum Linnaeus (1753: 95), Hypericum tetrapterum Fries (1823: 236), Silene nocturna Linnaeus (1753: 416), etc. Additional specimens examined:—GREECE. Peloponnisos: ca. 3.5 km S-SE of Sykea village, macchie and phrygana among olive groves, on flysch, 130 m, 36o43.970’ N, 22o57.079’ E, 06 May 2013, Kalpoutzakis 4294. ibid.: 29 July 2017, Kalpoutzakis 4959. ca. 2.5 km S-SE of Sykea village, macchie and phrygana among olive groves, on flysch, 120 m, 36o44.552’ N, 22o57.209’ E, 22 May 2017, Trigas 6312. Conservation:—In Neapoli Laconias, Petrorhagia laconica grows on a large sandy beach ca. 3.5 km long, between Neapoli and Viglafia village. The width of sand dunes along this beach ranges from 30 to 150 m, and the species is exclusively confined at the inner sand dune zone, where it is fairly rare. It usually forms small groups of 5–30 individuals, but single remote plants were also observed. The total population of Neapoli is estimated to include ca. 300–400 individuals, mainly concentrated at the eastern part of the beach. The Sykea population counts ca. 500 individuals distributed in an area of ca. 0.1 km2. Habitat loss and touristic development are the main threats that P. laconica faces at its locus classicus. Sand dunes have been partly turned into cultivated fields at the eastern half of the beach, almost certainly leading to a significant population decline. Species’ habitat also hosts thousands of swimmers during summer, being an unpredictable risk factor for its long term survival. The whole area, however, belongs to the Natura 2000 network of protected areas (Periochi Neapolis kai Nisos Elafonisos, GR2540002), and numerous human activities that could create additional risks for the population of P. laconica in Neapoli area are fortunately forbidden. Habitat loss is also the main threat that P. laconica faces in Sykea area, as more than 50% of the area previously covered with natural vegetation have been turned into cultivated fields. As a result, it is irrefutable that the species has suffered a severe population decline in the near past, which can be estimated as probably approaching the percentage of habitat loss. The flora of SE Peloponnisos has been intensively explored during the last decades (e.g. Kalpoutzakis & Constantinidis 2005, 2006, Greuter 2012, Kalpoutzakis et al. 2012). The recent discovery of P. laconica indicates that the species should not be common in this area. The habitat types that it grows (especially the mosaic with macchie and phrygana formations), however, are abundant in SE Peloponnisos. Consequently, the existence of additional populations of this tiny and easily overlooked species cannot be ruled out. The species’ extent of occurrence (EOO) does not exceed 25 km2 and since P. laconica is a very local species, its area of occupancy (AOO) is much narrower, apparently less than 1 km2. Ergo, due to: i) the restricted EOO and AOO, ii) the current existence of max. 900 mature individuals distributed in 2 populations and iii) the high possibility that the extant species’ localities and populations could be eradicated as a result of human interference, Petrorhagia laconica is assigned to the Endangered (EN) IUCN (2001) category, following criteria B1ab(i,ii,iii,iv)+2ab(i,ii,iii,iv). P. laconica should be carefully monitored, cultivated ex situ in botanical gardens and special conservation measures be taken to safeguard its populations. Taxonomic relationships:—Petrorhagia laconica undoubtedly belongs to Petrorhagia sect. Petrorhagia, as it is indicated by its 1-veined leaves, the presence of epicalyx bracts, the not abruptly clawed petals, the reticulate, blackish-brown seeds and the perennial life-cycle. The thickened seed margin is not a common feature of all species in P. sect. Petrorhagia, as it is indicated by Ball & Heywood (1964). P. fasciculata, P. thessala (Boiss.) P.W. Ball & Heywood (1964: 156), P. dianthoides (Sm.) P.W. Ball & Heywood (1964: 158) and P. grandiflora Iatrou (1985: 441) have flat, thin seed margins, similar to that of P. laconica. Within Petrorhagia sect. Petrorhagia, P. laconica shows close affinities to the members of subsect. Saxifragae. The solitary or paired flowers and the lanceolate, 1-veined epicalyx bracts advocate for the inclusion of P. laconica within subsect. Saxifragae. and seed morphological characters further support this taxonomic view. The simple or sparingly branched stems, however, resemble the members of subsect. Thessalae, which otherwise have significant morphological differences from P. laconica (e.g. capitate inflorescence, broadly ovate or sub-orbicular bracts, larger and different petals, etc.). P. laconica appears to be one of the most distinct species in P. sect. Petrorhagia. The results of multivariate morphometric analyses (Tables 2, 3, Figures 1, 2) clearly support its distinct taxonomic position within subsect. Saxifragae The most related species seem to be P. saxifraga and P. graminea, which, however, show significant morphological differences from P. laconica (see also Table 4). The possibility of vegetative reproduction and the tuberculate capsule have not been recorded in other Greek Petrorhagia species, and they probably represent unique features within the whole genus. The discovery of P. laconica is in line with Trigas et al. (2007, 2012), which state that SE Peloponnese is one of the important regions in Greece in terms of endemic species richness and conservation, as well as a significant diversity center for Petrorhagia, since more than 30% of the whole genus’ species diversity is hosted in this small region.

Acknowledgements

We are grateful to the curators of UPA and ATH for loaning specimens for our study. We also wish to thank Dr. Aimilia-Eleni Nikolopoulou (Agricultural University of Athens) for collecting specimens of Petrorhagia laconica with mature capsules.

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TABLE 1. Characters used in the morphometric analyses.

Character Detailed character definition abbreviation a1 Number of flowering stems a2 Length of flowering stems (cm) a3 Length of the unbrached part of flowering stem (cm) a4 Length of the inflorescence (cm) a5 Glandular hairs present at the lower part of the stem a6 Glandular hairs present at the middle part of the stem a7 Glandular hairs present at the upper part of the stem a8 Eglandular hairs present at the lower part of the stem a9 Eglandular hairs present at the middle part of the stem a10 Eglandular hairs present at the upper part of the stem a11 Number of flowers per flowering stem a12 Number of lateral branches per flowering stem a13 Length of lateral branches per flowering stem (cm) a14 Number of flowers per lateral branch a15 Number of flowers per terminal unit of the inflorescence a16 Length of lower leaves (mm) a17 Width of lower leaves (mm) a18 Length of middle leaves (mm) a19 Width of middle leaves (mm) a20 Length of internodes at the middle of the stem (mm) a21 Number of leaves at the middle of the stem a22 Ratio of leaves/internodes length at the middle of the stem a23 Number of veins on leaves a24 Length of petals (mm) a25 Width of petals (mm) a26 Length of calyx (mm) a27 Length of calyx teeth (mm) a28 Width of calyx teeth at their base (mm) a29 Length of pedicels (mm) a30 Bract length (mm) a31 Ratio pedicel length/calyx length a32 Ratio petal length/calyx length a33 Number of veins on sepals a34 Glandular hairs present on calyx a35 Eglandular hairs present on calyx a36 Epicalyx bracts present

TABLE 2. Stepwise discriminant analysis results for the five Petrorhagia species included in the present study based on fourteen morphological characters, as well as the discriminant functions’ eigenvalues and the proportion of variance (PV) explained by each discriminant function. DF1, DF2, DF3 and DF4 indicate the first, the second, the third and the fourth discriminant function, respectively. F, T and WD indicate the F-test P values, the tolerance values, as well as the Wilk’s lambda values of the discriminant functions and characters retained in the stepwise discriminant analysis, respectively. SML indicates the loadings from the stepwise discriminant analysis (i.e. the characters’ correlation with the discriminant functions). Character abbreviations follow Table 1. Higher values are shown in bold.

Character T F WD DF1 DF2 DF3 DF4 Eigenvalue - - - 205.23 27.22 3.12 1.38 PV - - - 86.6 11.5 1.3 0.6 WD - - - 0.00 0.00 0.10 0.42 SML a9 0.85 0.01 0.00 0.35 0.09 -0.06 0.11 a34 0.42 0.00 0.00 0.26 0.06 0.07 0.16 a15 0.58 0.00 0.00 -0.06 0.22 0.03 0.09 a35 0.51 0.00 0.00 -0.06 0.23 0.69 -0.23 a31 0.09 0.00 0.00 0.06 -0.03 -0.36 0.02 a8 0.45 0.00 0.00 0.20 0.03 0.34 0.32 a29 0.08 0.00 0.00 -0.09 -0.13 -0.29 0.03 a28 0.67 0.01 0.00 -0.06 -0.16 0.25 -0.14 a25 0.60 0.03 0.00 -0.07 -0.15 0.24 -0.02 a7 0.28 0.00 0.00 -0.05 0.18 -0.19 0.53 a6 0.16 0.00 0.00 -0.05 0.18 -0.19 0.53 a5 0.24 0.00 0.00 -0.09 0.38 -0.21 0.44 a14 0.38 0.00 0.00 -0.13 0.00 -0.08 0.37 a4 0.69 0.03 0.00 -0.15 0.00 0.02 0.28 a27 0.23 0.00 0.00 -0.05 -0.04 0.11 -0.26 a30 0.60 0.03 0.00 0.03 0.03 -0.04 -0.11

TABLE 3. Correct Leave-One-Out (LOO) cross-validation classification from the stepwise discriminant analysis. A, B, C, D and E indicate Petrorhagia fasciculata, P. graminea, P. phtiothica, P. saxifraga and P. laconica, respectively. PCC and MCC indicate the Proportional Chance Criterion and the Maximum Chance Criterion, respectively.

% Correct Taxon A B C D E Classification A 100.0 0.0 0.0 0.0 0.0 100.0 B 0.0 100.0 0.0 0.0 0.0 100.0 C 0.0 0.0 100.0 0.0 0.0 100.0 D 0.0 0.0 0.0 100.0 0.0 100.0 E 0.0 0.0 0.0 0.0 100.0 100.0 PCC < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 MCC - - - - < 0.001 Total - - - - 100.0

TABLE 4. Main morphological differences among Petrorhagia laconica, P. saxifraga and P. graminea.

Character P. laconica P. saxifraga P. graminea Underground Rooting at nodes Not rooting at nodes Not rooting at nodes stock Stems Glaucous-green, Green, arcuately ascending Green, sparingly procumbent to ascending to erect, usually branched branched, dichotomously or erect, simple or rarely to much-branched, divaricate, glabrous to with few [1–3 (–6)] diffusely paniculate, densely pubescent paniculate lateral branches glabrous to shortly at the upper 1/3, minutely pappilose or scabrid- pappilose-scabridulous pubescent at least in lower except the glabrous 1–4 part upper internodes Leaves Linear-subulate, rigid, Linear to linear-lanceolate, Linear, herbaceous, ± closely appressed to stem herbaceous, ± spreading spreading Inflorescence Flowers solitary or rarely Paniculate, flowers Dichotomously paired, 1 (–2) per solitary or in fascicles, 2– divaricate, flowers flowering stem, rarely up 59 per flowering stem solitary or in fascicles of to 6 and then inflorescence 2–3, 2–37 per flowering paniculate stem Epicalyx Present Present Absent bracts Calyx Minutely glandular Glabrous or occasionally Densely eglandular pubescent; ribs obscurely sparsely eglandular pubescent to glabrous; 1-veined, purplish-brown pubescent; ribs 3-veined, ribs 3-veined, green green Capsule 2.5–3 mm long, smooth or 3.5–5 mm long, smooth 3–5 mm long, smooth sparsely tuberculate at the lower half Seed 1.0–1.2 × 0.7–0.8 mm, 0.9–1.6 × 0.6–1.1 mm, 0.8–1.5 × 0.7–1.0 mm, ovate-oblong, with flat, ovate, with thick curved oblong to sub-orbicular, not thickened margins margins with slightly thickened, ± curved margins

Flowering May-August May-August September-January period

FIGURE 1. Stepwise discriminant analysis plot for the five Petrorhagia species. The squares indicate the function group centroid of each species.

FIGURE 2. Frequency distribution of discriminant scores for the first discriminant function (LD1). The numbers on the x axes represent the discriminant function values.

FIGURE 3. Petrorhagia laconica: (A) sand dunes northwest of Neapoli village in SE Peloponnisos, locus classicus, (B) habit, (C) middle part of stem with leaves, (D) upper part of stem with flower, (E) flower, side view, (F) flower, face view, (G) immature capsule, (H) seed, upper surface, (I) seed, lower surface.